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Patelia et al., 2:4
http://dx.doi.org/10.4172/scientificreports717
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Research Article
Sex Determination using Polymerase Chain Reaction
Emanual Michael Patelia*, Rakesh Thakur and Jayesh Patel
Department of Pharmaceutical Chemistry and Analysis, Indukaka Ipcowala College of Pharmacy, Gujarat, India
Abstract
The PCR is widely used technique in sex determination. PCR is also used for the determination of x linked
inherited disorders, with help of biopsied embryos into mothers. DNA polymerase enzyme replicates a piece of
DNA. Thus, chain reaction occurs and generating multiple copies of it. Polymerase chain reaction is frequently used
technique for sex determination. PCR can also be used for detecting the presence or absence of a particular piece
of DNA. In this method, we used PCR method for determination of the sex of three unknown bovine samples.PCR
techniques have developed to reduce the problems by increasing amplification quality.
Keywords: PCR; Sex determination; DNA polymerase
Introduction
Polymerase chain reaction is one type of the method used for the
determination of the sex. Polymerase chain reaction method (PCR)
was invented by Kary B. Mullis, they was also received the Nobel prize
in chemistry for PCR method invention on 10 December, 1993 [1].
PCR Method also used in other detection like mutations detecting,
monitoring cancer therapy, detecting bacterial, viral infection and also
sex determination [1].
Sex determination is one of the important methods, which help
to determine the sex of the developing embryo. Mostly x-y system is
use for the sex determination in human and mammals. Males have ‘x’
chromosome (xy) and female had both (xx) chromosomes. So with the
help of the chromosome sex is determined. It is not true for all the
organisms, for example in bird and reptiles male have homogametic
(ZZ) and female had heterogametic (ZW). Generally in male x and y
chromosome and in female xx chromosome.sex is determined by the
presence and absence of the y chromosome [2].
We can determine sex with the help of presence of unique sequence
on the y chromosome (SRY gene). The presence of SRY gene and
protein encoded is very important for testis differentiation in the early
embryo, if SRY gene is absent means it develops ovaries. The protein
of high mobility group (HMG) family is encoded with the help of SRY
gene. After that this protein triggers all the steps which are required to
development of male phenotype.
Taq DNA polymerase 2.4 μl. Finally, we prepared a 138 μl master mix.
After that we aliquot 23 μl of the master mix into 5 remaining tubes and
added 2 μl DNA to each tube. Then placed in the thermo cycler. In the
last PCR thermal cycle keep for initial denaturation at 95°C for 1 min,
annealing at 66°C for 1 min, extension at 72°C and final extension at
72°C for 3 min. Then for remaining use stored the samples at -20°C.
Gel electrophoresis
First Place the 16 sample comb in the MIDI Gel electrophoresis.
After that we poured gel solution (1.5% agarose) to a depth of 5 mm
and allow setting for 15 min. Then we add 5 μl of sample loading buffer
to each 0.2 ml tube from the PCR reaction. When the gel has set, take
the comb and add electrophoresis buffer (TAE) to cover the gel by
approximately 0.5 cm. Add 20 μl of all sample to individual well and
then run the gel at 100 V until the blue dye runs half way down the gel.
DNA quantification
DNA quantification helps to determine the quantity of amplifiable
DNA [3]. Quantities analysis is a procedure where the concentration
of a particular bimolecule in a sample is determined [4]. With the
help of a spectrophotometer the DNA and proteins are determine by
absorbance of UV light. At 260 nm absorbance DNA determined and
280 nm absorbance protein contamination was estimated. The blank
sample was used before measuring each sample absorbance.
Results
Material and Methods
The first lane in the figure above is marker sample.
DNA purification
Second lane is male
In the multiplex PCR method, In these unknown samples adding
buffer AL, BUFFER AW1 and buffer AW2 for isolation and purification
of DNA from unknown sample. After addition to each given sample
followed centrifugation. Then transfer the column into new 1.5 ml
tube than add buffer AE and incubated at room temperature and then
centrifuge. After that discard the column and leave the tube on ice for
PCR set up.
PCR
Third lane is female
Forth lane is sample 1
Fifth lane is sample 2
*Corresponding author: Emanuel M Patelia, Department of Pharmaceutical
Chemistry and Analysis, Indukaka Ipcowala College of Pharmacy, New Vallabh
Vidyanagar – 388121, Gujarat, India, E-mail: [email protected]
Received June 20, 2013; Published June 26, 2013
In these PCR method we used different reagent for the PCR
reaction like Buffer, primers,dNTPs, water and Taq DNA polymerase.
First we marked the label on six 0.2 ml tube as a ‘M’, ‘F’, ‘1’, ‘2’ , ‘3’
and ‘-’. Then we prepared a master mix in tube by added 15 μl PCR
buffer, dNTP mix 2.4 μl, primer mix 15 μl, sterile PCR water 103.2 μl,
Citation: Emanual MP, Rakesh T, Jayesh P (2013) Sex Determination using
Polymerase Chain Reaction. 2: 717 doi: 10.4172/scientificreports.717
Copyright: © 2013 Emanual MP, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Volume 2 • Issue 4 • 2013
Citation: Emanual MP, Rakesh T, Jayesh P (2013) Sex Determination using Polymerase Chain Reaction. 2: 717 doi: 10.4172/scientificreports.717
Page 2 of 4
Six lane is unknown sample 3
Seven lane is negative control
In the second lane, it is for male control. Ideally two bands should
be detected. One bands at the level of 218 bp and second band at 741
bp, First band for GAPDH gene and second band for SRY gene. In our
experiment result two week bands are visible (Figure 1).
Third lane is for female. There is only one band are visible at 218
bp, which is for GAPDH gene. In female there is no SRY gene. In our
experiment we detected week band at 218 bp in second lane.
Forth lane is for unknown sample 1. This sample was female, there
visible only one band at 218 bp and there is no SRY gene visible. In our
experiment one band is visible at 218 and no other bands are visible.
Fivth lane is for unknown sample 2. This sample was male, there
should two bands are visible, one band at 218 bp for housekeeping
GAPDH gene and second band at 741 bp level for SRY gene. In our
experiment one week band at 218 bp and no other band is visible.
Sixth lane is for unknown sample 3. This sample was male. There
should two bands are visible, one band at 218 bp for housekeeping
GAPDH gene and second band at 741 bp level for SRY gene. In our
experiment two bands are visible; first band at 218 bp for GAPDH and
second week bands at 741 bp for SRY gene.
Seventh lane is for negative control. There should be no band are
visible. In our experiment bright band is shown, which is some similar
to the primer dimer.
1. The terms regarding denaturing, annealing and extension refer
to in PCR.
Denaturing means heating of the PCR products upto 94°C for 30
sec, because of these hydrogen bonds between complementary
bases is broken and get single stranded DNA molecules.
Annealing means lowering the temperature at 66°C for 1 min
which are help in the binding of primers to single-stranded DNA
templates. Extension means heating of mixture to 72°C for 1 min
to which helps to DNA polymerase to synthesize a new DNA
strand complementary to DNA template.
2.Explanation regarding the role of GAPDH primers in this
experiment.
GAPDH genes are housekeeping genes. GAPDH primers were
provided to amplify these genes.
3. Explanation regarding the role of SRY primers in this experiment.
SRY gene which is sex determining region on Y chromosome.
For differentiation of testis in human embryo, the presence of
SRY gene and the protein encoded by it are necessary. If SRY
gene is absent, it results in development of ovaries SRY primers
were provided to amplify these genes (Table 1).
4. Calculation of GC content of each primer. Show your workings.
GC content= G+C/ A+T+G+C × 100
GAPDH F: 12/12+12 × 100 = 50
GAPDH R: 12/12+12 × 100 = 50
SRY F: 11/13+11 × 100 = 45.83
SRY R: 11/13+11 × 100 = 45.83
5. Calculation of the volume of each reagent in the master mix that
Figure 1: A positive PCR reaction for SRY should have an amplicon of 741bp, GAPDH should be 218bp.
The first lane in the figure above is marker sample.
Second lane is male
Third lane is female
Forth lane is sample 1
Fifth lane is sample 2
Six lane is unknown sample 3
Seven lane is negative control
Volume 2 • Issue 4 • 2013
Citation: Emanual MP, Rakesh T, Jayesh P (2013) Sex Determination using Polymerase Chain Reaction. 2: 717 doi: 10.4172/scientificreports.717
Page 3 of 4
DNA 4a
DNA 4b
DNA 4c
Abs 260
0.081
0.496
0.205
Abs 280
0.043
0.603
0.238
Ratio Abs 260/Abs 280
1.883
0.822
0.861
DNA concentration in the cuvette (μg DNA ml)
4.05
24.8
10.25
The dilution factor for the sample
49
49
49
DNA concentration in original sample (μg DNA ml)
198.45
1215.2
502.25
3.969
24.304
10.045
The amount of DNA in 20 μl sample (μg)
Table 1: List of components used for the calculations.
will be present in each PCR tube. Show your working.
primer, because of these base pairing even within the primers or pairing
between the primers, instead of other target sequence [1]. Because of
presence of large quantity of the primers, they encounter a partially
complementary molecule, rather than a perfectly complementary
template molecule, forming pairing .when binding are done produce a
fragment known as primer dimmers [1].
PCR buffer: 15/6 = 2.5
Dntp mix: 2.4/6 = 0.4
Primer mix: 15/ 6 = 2.5
Taq DNA polymerase: 2.4/6 = 0.4
6. Calculation for the final concentration of dNTPs present in each
PCR tube. Show your workings.
C1V1 =C2V2
50 Mm × 0.4 μL = C2 × 25 μL
C2 = 0.8 Mm
7. Which DNA sample has the highest concentration?
DNA 4b has the highest concentration (1215.2)
8. Which DNA sample is the most pure?
DNA 4a is the purest DNA with ratio of 1.883
9. Which DNA sample is the most suitable for use in PCR?
DNA4a
Discussion
There should be many errors possible at the time of the experiment
like at the time of the addition of the reagents, some reagent miss in the
master mix and some time possible errors with the concentration of the
reagent. Sometime large concentration of the magnesium also affect
to the DNA and presence of the protein. So we cannot get a specific
amplification [5]. Co-solvent and other additive like dimethyl sulfoxide
and formamide also affect to the polymarase chain reaction [5]. In
performing of the PCR, primer designing programs are very important
[6]. Because of not proper designed prime cannot get the proper result.
The most important affecting factor is selection of proper primers,
which affecting the polymerase chain reaction [7,8]. For the specific
amplification specific primers are requires otherwise do not match
to other target in certain orientations and in some area that allow
undesired amplification [8]. Sometime because of improper interaction
between the primer and template, efficiently errors occur and also
some contamination affect in the PCR reaction. 200-500bp is ideal
regions for amplification for analytical purpose [1]. Amplification
should be difficult to detect on agarose gel if region smaller than 200bp
and amplification should be not proper if strength is higher [1]. The
sequence of the primer is also very important [1]. Two primers same in
base composition and in length, it means two primers should be similar
annealing temperatures.
There are some factors which affect the design of the primers. Like
oligonucleotide made sequence with complementary itself or other
Role of SRY gene in gonadal development
Mammalian embryos have bipotential gonads, base on the paternal
and maternal chromosomal presence they can develop either into
a testis or an ovary [7]. Contribution of the x chromosome result in
ovarian differentiation and the y chromosome result in testicular
differentiation. At the time of development, bipotential gonad contains
pools of undifferentiated somatic cell precursors, which differentiate
into either testicular or ovarian [7].
On Y chromosome the gene for the transcription factory SRY is
located and it is responsible for take fate of testis in bipotential gonad.
There is SF1 factor (steroidogenic factor) called nuclear orphan
receptor. Which regulate immediate downstream gene called as SOX9
[7]. It is done with the help of the interaction on its testis specific
enhance. The role of the SOX9 is to control differentiation of precursor
cell into Sertoli cells. FGF9 and prostaglandins with the help of SOX9
promote testis development.
The process of the sex determination starts with differentiation of
somantic cells. The sequence on the DNA is observed when binding of
SRY to HMG box [7]. Helix loop helix (bHLH) transcription factors
have identified by scientists and they are known to monitor cell fate
specification, morphogenesis and differentiation. Early development of
embryo bHLH gene plays some role and affects a wide variety of tissues
[7]. Tcf21 is a one type of gene which helps in encodes transcription
factor for family bHLH. During male gonadal sex determination SRY
gene interacts with Tcf21, it is hypothesized and promotes somatic
cell differentiation. The polymerase chain reaction is one of the most
important technique in determining sex of given sample. PCR is very
useful for study and the analysis of genes [8].
References
1. Dale JW, van Schantz M, Plant N (2011) From Genes to Genomes: Concepts
and Applications of DNA Technology. Chichester, West Sussex: John Wiley &
Sons.
2. Khana P (2008) Cell and Molecular Biology. I. K. International Pvt Ltd.
3. Butler JM (2005) Forensic DNA Typing: Biology, Technology, and Genetics of
STR Markers. Elsevier Academic Press, London.
4. Langford A (2010) Practical Skills in Forensic Science. Pearson Prentice Hall,
Harlow, England.
5. Seviour RJ, Nielsen PH (2010) Microbial Ecology of Activated Sludge. IWA
Publishing.
6. You FM, Huo N, Gu YQ, Luo MC, Ma Y, et al. (2008) BatchPrimer3: a high
throughput web application for PCR and sequencing primer design. BMC
Bioinformatics 9: 253.
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Citation: Emanual MP, Rakesh T, Jayesh P (2013) Sex Determination using Polymerase Chain Reaction. 2: 717 doi: 10.4172/scientificreports.717
Page 4 of 4
7. Bhandari RK, Sadler-Riggleman I, Clement TM, Skinner MK (2011) Basic helixloop-helix transcription factor TCF21 is a downstream target of the male sex
determining gene SRY. PLoS One 6: e19935.
8. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, et al. (2012) PrimerBLAST: a tool to design target-specific primers for polymerase chain reaction.
BMC Bioinformatics 13: 134.
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